GB2217840A - Compositions for detection and/or quantification of an isolated acute phase protein in body liquids - Google Patents

Abstract

A diagnostic composition comprises phosphoryl choline residues and/or aminoethyl diydrogen phosphate residues chemically linked to an enzyme or a fluorescent agent or radioactive substance or a metal collold particle. Such compositions are used for quantitative and/or qualitative detection and/or determination of C-reactive protein in a test sample. The metal is preferably gold or silver.

Description

COMPOSITIONS FOR DETECTION AND/OR QUANTIFICATION OF AN ISOLATED ACUTE PHASE PROTEIN IN BODY LIQUIDS This invention relates to the detection and quantification of isolated C-reac-tive protein, which is an acute phase protein in body liquids.

C-reactive protein (herein called CRP) was first reported in 1930 as a human serum protein that binds to the C-polysaccharide of the pneumococcus cell membrane (Tillet W.S. and Francis T: "Seriological reaction in pneumonia with a non-protein somatic fraction of pneumococcus", J. Exp. Med. 52: 561-571, 1930). Later it was demonstrated that C-reactive protein binds to phosphoryl choline residues and to aminoethyldihydrogen phosphate residues of this fraction, and these bindings have been utilized for the purification of C-reactive protein from serum. (Volanakais J.E. de al: J. Immunol. Methods 23, 285-295, 1978, and Potent M. & al: FEBS Lett. 88, 172-175, 1978).

Later it was discovered that CRP concentrations rise dramatically during pneumococcus infections and several forms of infectious inflammations (Abernathy TS and Avery OP: "The occurrence during acute infections of a protein not normally present in the blood", J. Exp. Med. 73: 173-182, 1941). This non-specific response to bodily injury was classified as a part of the "acute-phase response", in which changes in the concentrations of serum proteins parallel the course of inflammation or tissue injury.

Although their exact roles remain unclear, CRP and the other acute-phase proteins function as mediators, inhibitors or participants in the process of inflammation. CRP is a trace constituent in blood, where serum levels in healthy adults normally remain below 5 mg/l. The serum concentration of CRP rises rapidly after onset of infections, inflammations and tissue injury, and falls rapidly to normal levels as healing or recovery occurs (Whicher JT, Bell AM and Southall PJ: "Inflammation: Measurement in clinical management", Diagnostic Med. 81:62, 1981). As a clinical tool CRP quantitation appears to be more useful than the standard indices of inflammation5 such as fever, erythrocyte sedimentation rate and leucocyte count.The magnitude of the increase from normal to acute inflammatory concentration also contributes to the utility of CRP in monitoring disease activity (Pepys MB: "C-reactive-protein 50 years on; Lancet 1: 653-657, 1981).

The CRP molecule consists of 5 identical subunits held together by non-covalent bonds (Pepys MB: see above). Several methods for CRP detection and quantification are available, including immunoprecipitation (Andersen HC and McCarty M: "Determination of C-reactive protein in blood as measure of the activity of the disease process in acute rheumatic fever", Am. J. Med. 8: 445-445, 1950, and Wadsworth C: "A rapid spot immuno-precipitate assay method applied to quantitating C-reactive protein in paediatric sera". Scand. J. Immunol. 6: 1263-1272, 1977); latex agglutination (Singer JM, Potz CM, Pader E and Elster SK: "The latex agglutination test". Am J. Clin. Pathol 28: 611-617, 1957); radial immunodiffusion (Nilsson LA: "Comparative testing of precipitation methods for quantitation of C-reactive protein in blood serum".

Acta Pathol. Microbiol. Scand. 73: 129-144, 1968); electroimmunodiffusion (Gil CW, Fisher CL, and Holleman CL: "A rapid method for protein quantitiation by electroi mmunodiffusion", Clin. Chem. 17: 501-504, 1971); radioimmunoassay (Claus DR, Osman AP, and Gewurz H: "Radioimmuniassay of human C-reactive protein and labels in normal sera".

3. Lab. Clin. Med. 87: 127-128, 1976 and Shine B, DeBeer FC, and Pepys MD: "Solid phase radioimmunoassay for human C-reactive protein".

Clin. Chem. Act 117: 13-23, 1981); nephelometry (Deaton CD, Maxwell KW, Smith RF and Crevelling RL: "Use of laser nephelometry in the measurement of serum proteins". Clin. Chem. 22: 1465-1471, 1976 and Gil CW, Bush WS, Burleigh WM, and Fisher CL: "An evaluation of a Creactive protein assay using a rate immunonephelometric procedure".

AJCP 75: 50-55, 1981); fluoroimmunoassay (Anne L, Eimstad W, Bellet N, and Fisher C: "Development of homogeneous fluorescent rate immunoassay for C-reactive protein". Clin. Chem. 27: 1075, 1981 and Ullman F, Schwarzberg M and Rubensten KE: "Fluorescent excitation transfer immunoassay: A general method for the determination of antigens", J. Biol. Chem. 251: 4172-4178, 1976); and enzyme-labelled immunoassay (Gibbons I, Skiold C, Rowley GL and Ullman EF: "Homogeneous enzyme immunoassay for proteins employing beta-galactosidase".

Anal. Biochem. 102: 167-170, 1981).

CRP is among the plasma proteins known to have the most prominent concentration difference between the normal plasma concentration and the plasma concentration in acute inflammatory and infectious-phase reactions.

Since the increase in blood concentration of CRP is a very rapid response to infections and tissue damage, its determination is especially valuable in acute medical situations. A large number of acute medical incidents take place in general medical practice far from advanced medical laboratories.

Most of the known methods for the detection and/or quantification of increased CRP utilize the laboratory equipment like spectrophotometers, radioactivity counters and nephelometric equipment, and a time-consuming isolation of serum or plasma from whole blood is necessary. Simple qualitative latex agglutination techniques do not utilize advanced equipment, but isolation of serum and/or plasma is necessary. A fast and simple method to detect and/or quantify acute-phase response of blood proteins in whole blood is thus needed.

All methods used in clinical practice for CRP in plasma or serum utilize immunoglobulins with specific affinity for these proteins. Such immunoglobulins may be of polyclonal or monoclonal origin. Polyclonal antibodies with specific affinity for CRP are obtained by immunization of animals with purified antigens. Monoclonal antibodies reactive to the said antigen may be obtained by fusion of spleen cells from animals immunized with antigen or fragments thereof with suitable cancer cell lines (Khler and Milstein. "Continuous cultures of fused cells secreting antibodies of predefined specificity", Nature 286, 495-497, 1975). In vivo immunization techniques may also be applied. Several of the immunological techniques utilize modified antibodies, i.e. antibodies conjugated to enzymes or fluorescent residues or radioactive labels. Such modifications and conjugations may be obtained by numerous different known methods, as described by O'Sullivan MJ, Bridges JW and Marks V: "Enzyme immunoassay: A review", Annals of Clinical Biochemistry, 1979, 16, 221240.

The present invention provides a diagnostic composition comprising phosphoryl choline residues and/or aminoethyl dihydrogen phosphate residues chemically linked to an enzyme or a fluorescent agent or radioactive substance or a metal colloid particle. If a metal colloid is used, it may be gold or silver.

In accordance with the present invention, the compositions are used for quantitative and/or qualitative detection and/or determination of Creactive protein in a test sample.

Thus, in the present invention, phosphoryl choline residues and/or aminoethyl dihydrogen phosphate residues, bound to a signal substance, are used for the detection/quantitation of C-reactive protein. These ligands bind to CRP in the presence of calcium. The measurement of the conjugated signal molecule after removal of not-bound signal substance is used for detection/quantitation of CRP in a test sample, usually from body liquids or mixtures of body liquids and other reagents. Depending upon the nature of the signal molecule, the detection system may consist of a spectrophotometer, fluorometer, radioactivity ratemeter or the naked eye.

The phosphoryl choline (PC) residues or aminoethyl-dihydrogenphosphate (AEDP) residues, which are bound to different signal molecules, are ligands that bind CRP in the presence of Ca++ ions. CRP consists of five identical subunits non-covalently associated with each other in a disclike configuration. Each subunit of CRP is able to bind to both the amino and the phosphate groups of both the PC and the AEDP residues. The binding of CRP to PCT and AEDP is dependent upon the presence of Ca++ ions in solution: usually amounts of 1-2 mmol/l are sufficient. The exact position of Ca in the binding structure has not yet been fully disclosed, but the binding is prevented or broken by the addition of an excess of strong Cabinding substances such as EDTA (ethylenediaminetetra-acetic acid).

Since the five subunits of CRP are identical it is possible for several PC or AEDP units to become bound to the same CRP molecule. In this way it is possible to use the binding to PC or AEDP for isolation of CRP from other plasma proteins by using immobilised PC and/or AEDP residues. After eventually washing away other constituents of a test sample, it is possible to use a second PC or AEDP residue linked to a signal substance for quantitative or qualitative determination of the isolated CRP. The amount of bound signal substance will be in proportion with the level of CRP in the test sample. It is further possible to use conjugates based upon polymeric derivates of PC or AEDP residues, since the CRP molecule has several binding sites for these ligands.

Due to the nature of CRP5 it is possible to use a binding principle for isolation of CRP that is different from the principle binding the signal molecule to CRP.

According to this, the use of immobilised antibodies to CRP, monoclonal or polyclonal, for isolation of CRP prior to the attachment of the labelled signal molecule, is an attractive approach.

The present invention describes conjugates intended to bind to already immobilised CRP. This immobilisation of CRP may be constituted by a solid phase made of a porous membrane containing CRP-binding substance, further attached to a support designed for a dipstick test.

Another approach is to use polymeric surfaces as plastic tubes, cuvettes or 96-well microtitre plates as support for the immobilised CRP-binding molecule.

The conjugates may consist of PC or AEDP residues covalently attached to enzymes such as alkaline phosphatase or peroxidase. The binding may either be directly between the ligand and the protein or by a linkage. The CRP will be isolated by binding to a solid support and the added conjugate will become attached to the CRP in proportion to the amount of CRP In the test sample. By adding substrates for the enzymes, the measured enzyme activity will give a quantitative or qualitative measurement of CRP level in the test sample. If designed as a dip-stick, it is convenient to use a substrate giving a coloured insoluble product. Thus the intensity of colour developed on the dip-stick indicates the level of CRP. Performed in a tube, cuvette or in wells, it is convenient to use a substrate giving a coloured soluble product.The amount of a soluble product formed, and thus the amount of CRP isolated, may be measured by photometric devices.

Alternatively, the PC or AEDP residues may be covalently attached to a fluorescent agent such as fluorescamine or o-phthalaldehyde. As an example, this may be achieved by coupling the fluorescent agent to the amino group of aminophenylphosphoryl-choline or to the amino group of aminoalkylaminoethyl dihydrogen phosphate. By introducing these conjugates to the immobilised CRP and washing away not-bound fluorescent conjugate, the measurement of fluorescence will indicate the level of CRP.

By using colloidal particles of gold or silver, a signal substance is created when these particles are coated with PC or AEDP residues. This is achieved either by direct coupling of ligands to the particle surface or by adsorbing CRP-binding ligand-containing protein on the colloidal particle.

The colloid is itself coloured and therefore it could be used directly as a signal substance.

Applied to a porous membrane containing isolated CRP, the colloid will be bound to the membrane in proportion to the CRP. After removal of not-bound colloid, the colour intensity could be monitored directly and the amount of bound CRP estimated.

A fourth alternative is to use a radioactive nucleide as a signalgenerating substance. The radioactive substance may be linked to the binding ligands directly or it may be first bound to a larger molecule as a protein, before the protein is linked to the CRP-binding ligand. The radioactive conjugate will be presented to the immobilised CRP and excess of conjugate removed by washing.

Measurement of the degree of bound radioactivity by the use of scintillation counting will be in direct correlation with the amount of the isolated/immobilised CRP.

Claims (5)

1. A diagnostic composition comprising phosphoryl choline residues and/or aminoethyl dihydrogen phosphate residues chemically linked to an enzyme or a fluorescent agent or radioactive substance or a metal colloid particle.

2. A composition according to Claim 1 in which the phosphoryl choline residues and/or aminoethyl dihydrogen phosphate residues are chemically linked to the said signal substances by means of molecules different from the PC and/or AEDP residues and different from the enzyme, fluorescent agent, radioactive substance and/or metal colloid.

3. A composition according to Claim 1 and 2 in which the phosphoryl choline residues and/or aminoethyl dihydrogen phosphate residues are present in a polymeric form, with or without other molecules between the monomeric units of the phosphoryl choline residues and/or aminoethyl dihydrogen phosphate residues.

4. A composition according to any one of Claims 1 to 3 in which the metal colloid is gold or silver.

5. The use of a composition according to any one of Claims 1 to 4 for quantitative and/or qualitative detection and/or determination of Creactive protein in a test sample.